Hi, I can't see the figure but if you see TBR1 outside of the nucleus, it is most likely due to non-specific binding of the secondary. Have you tried blocking with more serum and for longer? I also would recommend that you use an isotype control to confirm the non-specific binding.

Thank you very much. Do you think donkey serum is fine?? and how long would you recommend.

Thank you very much!!

Donkey serum is fine only if your secondary is raised in Donkey. So do check your secondary antibody that you use. I would normally use at least 10% serum in my block.

Check out some good tips on ICC/IF procedures in the attached link. Good luck.

http://www.abcam.com/protocols/immunocytochemistry-immunofluorescence-protocol

Thank you very much